Hypoglycemia remains the major factor limiting the use of intensified insulin therapy that has been shown to prevent complications in type 1 diabetes (T1DM). This project continues studies focused on the mechanisms that trigger glucose counterregulation and lead to the development of hypoglycemia-associated autonomic failure and hypoglycemia unawareness in patients with T1DM. The protocols rely, where possible, on human investigation, but also utilize unique rodent models to test specific hypotheses. We will evaluate the molecular mechanisms used by glucose-sensing neurons involved in the activation of counterregulatory responses. Specifically, we test the hypothesis that KATP channels play an important role. In these studies, we will modulate the activity of KATP channels in putative brain glucose-sensing regions (e.g. VMH) of awake rats during hypoglycemia and study hypoglycemic counterregulation in awake KIR 6.2 knockout mice using the glucose clamp technique. The role of other putative molecules in glucose sensing will also be examined (e.g., glucokinase and AMP-kinase). To determine the mechanisms responsible for hypoglycemia-associated autonomic failure (HAAF), we will evaluate the contribution of corticotropin-releasing hormone (CRH) to HAAF in a rodent model using CRH receptor blockade. In addition, we will attempt to resolve the relative contribution of the individual components of the hypothalamic-pituitary-adrenal axis to HAAF in humans by determining whether HAAF can develop in the setting of CRH stimulation and blockade of cortisol secretion. We will examine the effect of brief and long-term exposure to recurrent antecedent hypoglycemia on cognitive function and corresponding changes in local brain glucose metabolism in rats during euglycemia and hypoglycemia using a hippocampus-dependent spatial working memory task. Brain glucose and monocarboxylic acid transporters as well as glucocorticoid and mineralocoticoid receptors will be assessed, as well. Finally, we will prospectively determine whether islet transplantation that restores normoglycemia in T1DM patients with severe hypoglycemia unawareness has an impact on cognitive performance during eu- and hypoglycemia as assessed by fMRI. We will also evaluate sympathoadrenal responses in these patients before and at 3 and 12 months after islet transplantation. The long-term goal is to develop novel clinical strategies aimed at minimizing the risk of insulin-induced hypoglycemia in patients with T1DM.